Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
  • C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic Table
  • C07F9/02—Phosphorus compounds
  • C07F9/547—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom
  • C07F9/6524—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom having four or more nitrogen atoms as the only ring hetero atoms
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
  • C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
  • C07H1/00—Processes for the preparation of sugar derivatives
  • C07H1/02—Phosphorylation
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
  • C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic Table
  • C07F9/02—Phosphorus compounds
  • C07F9/06—Phosphorus compounds without P—C bonds
  • C07F9/08—Esters of oxyacids of phosphorus
  • C07F9/09—Esters of phosphoric acids
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
  • C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic Table
  • C07F9/02—Phosphorus compounds
  • C07F9/06—Phosphorus compounds without P—C bonds
  • C07F9/16—Esters of thiophosphoric acids or thiophosphorous acids
  • C07F9/165—Esters of thiophosphoric acids
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
  • C07H1/00—Processes for the preparation of sugar derivatives
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
  • C07H23/00—Compounds containing boron, silicon or a metal, e.g. chelates or vitamin B12

Definitions

• the present invention relates to a phosphoramidite activator, a method for activating phosphoramidite using the activator, and a method for synthesizing a phosphate ester or a thiophosphate ester.
• Nucleic acid which is a general term for deoxyribonucleic acid (DNA) and ribonucleic acid (RNA), is a type of biopolymer having a structure in which nucleotides, which are constituent units, are linked in a chain by a phosphate ester bond (phosphodiester bond). .. Chemical synthesis of nucleic acids (artificial synthesis) is an important technique in biochemistry, and development of high-yield and high-purity nucleic acid synthesis methods is required.
• Nucleic acid synthesis usually involves a series of reaction cycles, including (A) deprotection of nucleosides or oligonucleotides, (B) coupling reactions, (C) capping of unreacted products, and (D) oxidation or sulfide reactions of coupling products.
• the nucleic acid chain is extended by repeating the above procedure.
• a "solid-phase synthesis method” that is quick, simple, and can be automated is often adopted.
• (B) nucleic acid synthesis by the "phosphoroamidite method” using phosphoramidite in the coupling reaction has become the mainstream. Specific examples of the reaction cycle in the phosphoramidite method are shown below.
• Ac represents an acetyl group
• Base represents a nucleobase selected from adenine, guanine, cytosine, thymine and uracil
• DMTr represents a 4,4'-dimethoxytrityl group
• iPr represents an iPr.
• A represents a tert-butyldimethylsilyl group
• M represents an oxygen atom or a sulfur atom.
• Acetonitrile is generally used as a solvent in nucleic acid synthesis by the phosphoramidite method.
• the above-mentioned BTT has poor solubility in acetonitrile, and in a high concentration BTT-acetonitrile solution, BTT crystals are precipitated at a low temperature, which may clog the piping of the nucleic acid synthesizer. Therefore, although phosphoramidite can be activated more quickly and efficiently if the concentration of BTT in acetonitrile is relatively high, the concentration that can be stored and used as a BTT-acetonitrile solution is 0.25 mol / L. It is restricted.
• Patent Document 1 discloses a phosphoramidite activator to which N-alkylimidazole is added as a co-solvent.
• the phosphoramidite activator described in Patent Document 1 described above is composed of an additive-free BTT-acetonitrile solution, although the solubility of BTT in acetonitrile is improved by adding N-alkylimidazole.
• the present invention has been made in view of such circumstances, and an excellent phosphoramidite activator that solves the above-mentioned problems, a method for activating phosphoramidite using the activator, and phosphorus.
• An object of the present invention is to provide a method for synthesizing an acid ester or a thiophosphate ester.
• the present inventors have added at least one compound selected from the group consisting of piperidine, pyrrolidine, N-alkylpiperidine and N-alkylpyrrolidine to a BTT-acetoform solution to add BTT to acetonitrile.
• a BTT-acetoform solution to add BTT to acetonitrile.
• the present invention includes the following inventions [i] to [xi].
• Amidite activator hereinafter, may be abbreviated as the phosphoramidite activator of the present invention).
• the molar concentration of N-alkylpiperidin in (i) is 13.7% or more with respect to the molar concentration in (ii), and the molar concentration of N-alkylpyrrolidine in (i) is (i).
• the molar concentration of piperidine (i) is 17.0% or more with respect to the molar concentration of (ii), and the molar concentration of pyrrolidine of (i) is 24.0 with respect to the molar concentration of (ii).
• the phosphoromidite activator according to any one. [Vii] A method for activating phosphoramidite, which comprises a step of reacting phosphoramidite with the phosphoramidite activator according to any one of the above inventions [i] to [vi] (hereinafter, the present invention). It may be abbreviated as the activation method of.).
• [Ix] Phosphite by reacting phosphoramidite with nucleoside or an oligonucleotide in the presence of the phosphoramidite activator according to any one of the above inventions [i] to [vi].
• a method for synthesizing a phosphoric acid ester or a thiophosphate ester which comprises a step of producing an ester and (2) a step of oxidizing or sulfurizing the phosphite ester to produce a phosphoric acid ester or a thiophosphate ester (hereinafter referred to as the present invention). It may be abbreviated as the method for synthesizing the invention).
• the phosphoramidite is a compound represented by the following general formula (I)
• the phosphoric acid ester is a compound represented by the following general formula (II-1)
• the thiophosphate ester is the following general formula.
• 1 represents a nucleoside and A 2 represents a nucleoside or an oligonucleotide. ].
• the phosphoramidite activator of the present invention comprises at least one compound selected from the group consisting of (i) piperidine, pyrrolidine, N-alkylpiperidine and N-alkylpyrrolidine, (ii) 5-benzylthio-1H-tetrazole, and. (iii) It contains acetonitrile.
• alkyl group in the N-alkylpiperidine and N-alkylpyrrolidine of (i) examples include alkyl groups having 1 to 6 carbon atoms, and among them, those having 1 to 3 carbon atoms are preferable.
• the alkyl group may be linear, branched or cyclic, and is preferably linear or branched. Specific examples thereof include a methyl group, an ethyl group, an n-propyl group, an isopropyl group, an n-butyl group, an isobutyl group, an n-pentyl group, an isopentyl group, an n-hexyl group, an isohexyl group and the like. , Ethyl group, n-propyl group, isopropyl group are preferable, and methyl group is particularly preferable.
• N-alkylpiperidine of (i) examples include N-methylpiperidine, N-ethylpiperidine, Nn-propylpiperidine, N-isopropylpiperidine, Nn-butylpiperidine, N-isobutylpiperidine, and Nn-pentyl.
• Examples thereof include piperidine, N-isopentyl piperidine, Nn-hexyl piperidine, N-isohexyl piperidine, N-methyl piperidine, N-ethyl piperidine, Nn-propyl piperidine, N-isopropyl piperidine, and N-methyl piperidine. Especially preferable.
• N-alkylpyrrolidine (i) examples include N-methylpyrrolidine, N-ethylpyrrolidine, Nn-propylpyrrolidine, N-isopropylpyrrolidine, Nn-butylpyrrolidine, N-isobutylpyrrolidine, and Nn-pentyl.
• examples thereof include pyrrolidine, N-isopentylpyrrolidine, Nn-hexylpyrrolidine, N-isohexylpyrrolidine, etc., preferably N-methylpyrrolidine, N-ethylpyrrolidine, Nn-propylpyrrolidin, N-isopropylpyrrolidine, and N-methylpyrrolidin Especially preferable.
• (i) may contain only one of piperidine, pyrrolidine, N-alkylpiperidine and N-alkylpyrrolidine alone or in combination of two or more.
• any one compound selected from the group consisting of piperidine, pyrrolidine, N-alkylpiperidine and N-alkylpyrrolidine is preferable; it is selected from the group consisting of piperidine, N-alkylpiperidine and N-alkylpyrrolidine. Any one compound is more preferred; N-alkylpiperidine or N-alkylpyrrolidine is even more preferred; N-methylpiperidine or N-methylpyrrolidine is even more preferred.
• the lower limit of the molar concentration of (ii) is usually 0.20 mol / L or more, preferably 0.25 mol / L or more, and 0.30 mol / L or more with respect to the total volume of the phosphoramidite activator of the present invention. More preferable.
• the upper limit of the molar concentration of (ii) is usually 1.00 mol / L or less, preferably 0.50 mol / L or less, and 0.40 mol / L or less with respect to the total volume of the phosphoramidite activator of the present invention. More preferable.
• the lower limit of the molar concentration of piperidine in (i) is preferably 17.0% or more with respect to the molar concentration of (ii); the upper limit is usually 150% or less with respect to the molar concentration of (ii). It is preferably 100% or less, more preferably 50.0% or less.
• the lower limit of the molar concentration of pyrrolidine in (i) is preferably 20.0% or more, more preferably 24.0% or more with respect to the molar concentration of (ii); the upper limit is the molar concentration of (ii). It is usually 150% or less, preferably 100% or less, and more preferably 50.0% or less.
• the lower limit of the molar concentration of N-alkylpiperidine in (i) is preferably 13.7% or more, more preferably 19.3% or more with respect to the molar concentration of (ii); the upper limit is the molar concentration of (ii). It is usually 150% or less, preferably 100% or less, and more preferably 50.0% or less.
• the lower limit of the molar concentration of N-alkylpyrrolidine in (i) is preferably 16.0% or more, more preferably 19.3% or more with respect to the molar concentration of (ii); the upper limit is the molar concentration of (ii). It is usually 150% or less, preferably 100% or less, and more preferably 50.0% or less.
• the phosphoromidite activator of the present invention includes (i) at least one compound selected from the group consisting of piperidine, pyrrolidine, N-alkylpiperidine and N-alkylpyrrolidine, and (ii) 5-benzylthio-1H-tetrazole. And (iii) may contain components other than acetonitrile, but those consisting only of (i), (ii) and (iii) are preferred; among them, (i) piperidine, pyrrolidine, N-alkylpiperidine and N.
• any one compound selected from the group consisting of alkylpyrrolidines (ii) 0.25 mol / L or more 5-benzylthio-1H-tetrazole, and (iii) acetonitrile alone;
• piperidine It consists of only one compound selected from the group consisting of pyrrolidine, N-alkylpiperidine and N-alkylpyrrolidine, (ii) 0.25 mol / L or more 5-benzylthio-1H-tetrazole, and (iii) acetonitrile.
• the molar concentration of piperidine in i) is 17.0% or more with respect to the molar concentration in (ii)
• the molar concentration of pyrrolidine in (i) is 20.0% or more with respect to the molar concentration in (ii)
• (i) The molar concentration of N-alkylpiperidine in (ii) is 13.7% or more with respect to the molar concentration of (ii)
• the molar concentration of N-alkylpyrrolidine in (i) is 16.0% or more with respect to the molar concentration of (ii).
• the molar concentration of N-alkylpiperidin or N-alkylpyrrolidine in (i) is 19.3% or more with respect to the molar concentration in (ii);
• N-alkylpiperidin or It consists only of N-alkylpyrrolidin,
• 5-benzylthio-1H-tetrazole of 0.30 mol / L or more and
• acetonitrile and the molar concentration of N-alkylpiperidine or N-alkylpyrrolidin in (i) is (ii).
• the phosphoramidite activator of the present invention may be prepared by mixing (i), (ii) and (iii) according to a method known per se. Specifically, for example, it is prepared by appropriately setting the amounts of (i) and (ii) to be used so as to obtain the desired molar concentration, first mixing (i) and (iii), and then mixing (ii). Can be done.
• the preparation of the phosphoramidite activator of the present invention is not particularly limited as long as the preparation conditions (temperature, pressure, atmosphere, etc.) can be mixed without affecting (i), (ii) and (iii).
• it may be prepared at 15 to 40 ° C., normal pressure (1 atm), and an atmosphere of an inert gas (for example, nitrogen, argon, etc.).
• the activation method of the present invention comprises the step of reacting phosphoramidite with the phosphoramidite activator of the present invention.
• the phosphoramidite in the activation method of the present invention is a monoamide derivative of a phosphorous acid diester, and in the present invention, in particular, a nucleoside phosphoramidite in which a nucleoside is bound to one ester portion of the phosphorous acid diester. It shall indicate that.
• the phosphoramidite in the activation method of the present invention is not particularly limited as long as it is usually used in this field, and a commercially available product or a product appropriately synthesized by a method known per se may be used.
• the nucleoside in phosphoramidite in the activation method of the present invention is a compound composed of a nucleobase and a sugar, and it is preferable that the hydroxy group (5'-OH group) at the 5'position is protected by a protecting group.
• the nucleobase include adenine, guanine, cytosine, thymine and uracil, and derivatives in which a part thereof is substituted or modified.
• Specific examples of the sugar include ribose and deoxyribose, and derivatives in which a part thereof is modified.
• the protecting group includes an acetyl group, a tert-butyldiphenylsilyl group, a tert-butyldimethylsilyl group, a 4,4'-dimethoxytrityl group (4,4'-dimethoxytriphenylmethyl group), and a 4-monomethoxytrityl group. (4-Methoxytriphenylmethyl group), and a 4,4'-dimethoxytrityl group is preferable.
• the nucleoside in phosphoramidite in the activation method of the present invention has a hydroxy group (2'-OH group) at the 2'position, the 2'-OH group is also a protecting group like the 5'-OH group. Those protected by are preferable.
• the protecting group examples include an acetyl group, a tert-butyldiphenylsilyl group, a tert-butyldimethylsilyl group, a triisopropylsiloxymethyl group, a 4,4'-dimethoxytrityl group and a 4-monomethoxytrityl group, and tert- A butyldimethylsilyl group is preferred.
• the nucleoside in the phosphoramidite in the activation method of the present invention is one in which functional groups other than the 5'-OH group or the 2'-OH group are protected as necessary so as not to cause a side reaction. Is preferable.
• the amino group in the nucleobase is an acetyl group, an isobutyryl group, a benzoyl group, 4 -(tert-Butyl) It is preferable that it is protected by an acyl group such as a benzoyl group or a phenoxyacetyl group.
• R 1 represents a group selected from the following functional group group (I-1): (In the functional group group (I-1), n 1 and n 2 each independently represent an integer of 1 to 6.), R 2 and R 3 each independently represent an alkyl group having 1 to 6 carbon atoms, and R 2 and R 3 may be bonded to each other to form a 5- to 7-membered heterocyclic structure.
• 1 represents a nucleoside.
• n 1 and n 2 in the functional group group (I-1) of the general formula (I) integers of 2 to 5 are preferable, and 2 is more preferable.
• a cyanoethyl group is particularly preferable.
• the alkyl group having 1 to 6 carbon atoms in R 2 and R 3 of the general formula (I) those having 2 to 3 carbon atoms are preferable.
• the alkyl group may be linear, branched or cyclic, and is preferably branched. Specifically, for example, methyl group, ethyl group, n-propyl group, isopropyl group, n-butyl group, isobutyl group, n-pentyl group, isopentyl group, cyclopentyl group, n-hexyl group, isohexyl group, cyclohexyl group and the like.
• the ethyl group, n-propyl group, and isopropyl group are preferable, and the isopropyl group is particularly preferable.
• the heterocyclic structure of the 5- to 7-membered ring is R 2 and R 3 .
• the nitrogen atom in the bonded general formula (I) it may further have one or more heteroatoms.
• Specific examples of the 5- to 7-membered ring heterocyclic structure include pyrrolidine, piperidine, hexamethyleneimine, oxazolidine, morpholine, thiazolidine, thiomorpholine and the like, with piperidine, morpholine and thiomorpholine being preferred.
• R 2 and R 3 of the general formula (I) an alkyl group having 2 to 3 carbon atoms is preferable, and an isopropyl group is particularly preferable. Further, it is preferable that R 2 and R 3 of the general formula (I) are the same.
• the content of (ii) 5-benzylthio-1H-tetrazole in the phosphoramidite activator of the present invention is usually 1 with respect to 1 mol of phosphoramidite. It is an amount of up to 20 equivalents.
• the activation method of the present invention is not particularly limited as long as the reaction conditions (temperature, pressure, atmosphere, etc.) in which the phosphoramidite and the phosphoramidite activator of the present invention can react without delay are not particularly limited, and for example, 10 to 40 ° C. , Normal pressure, inert gas atmosphere.
• the reaction time in the activation method of the present invention cannot be unequivocally determined because it can change depending on the reaction conditions, but it is usually 1 minute to 3 hours.
• the synthetic method of the present invention is (1) a step of reacting phosphoramidite with a nucleoside or an oligonucleotide in the presence of the phosphoramidite activator of the present invention to produce a phosphite ester, and (2). It includes a step of oxidizing or sulfurizing the phosphite ester to produce a phosphoric acid ester or a thiophosphate ester.
• the phosphoramidite in the step (1) in the synthesis method of the present invention includes the same phosphoramidite as the phosphoramidite in the activation method of the present invention, and the preferred ones are also the same.
• the nucleoside in the step (1) in the synthetic method of the present invention is not particularly limited as long as it is a natural or artificial nucleoside usually used in this field, and a commercially available nucleoside or a nucleoside appropriately synthesized by a method known per se may be used. Good.
• the nucleoside in the step (1) in the synthetic method of the present invention is a compound composed of a nucleobase and a sugar, and it is preferable that the hydroxy group (3'-OH group) at the 3'position is protected by a protecting group.
• the nucleobase include adenine, guanine, cytosine, thymine and uracil, and derivatives in which a part thereof is substituted or modified.
• Specific examples of the sugar include ribose and deoxyribose, and derivatives in which a part thereof is modified.
• the protecting group examples include an acetyl group, a tert-butyldiphenylsilyl group, a tert-butyldimethylsilyl group, a 4,4'-dimethoxytrityl group and a 4-monomethoxytrityl group, and a tert-butyldimethylsilyl group is preferable. ..
• the nucleoside in the step (1) of the synthetic method of the present invention has a 2'-OH group, it is preferable that the 2'-OH group is also protected by a protecting group like the 3'-OH group.
• the protecting group examples include an acetyl group, a tert-butyldiphenylsilyl group, a tert-butyldimethylsilyl group, a triisopropylsiloxymethyl group, a 4,4'-dimethoxytrityl group and a 4-monomethoxytrityl group, and tert- A butyldimethylsilyl group is preferred.
• the nucleoside in the step (1) in the synthetic method of the present invention preferably protects functional groups other than the 3'-OH group or the 2'-OH group as necessary so as not to cause a side reaction. ..
• the amino group in the nucleobase is an acetyl group, an isobutylyl group, a benzoyl group, 4 -(tert-Butyl)
• a benzoyl group, a phenoxyacetyl group or the like protected with an acyl group is preferable.
• the oligonucleotide in the step (1) in the synthesis method of the present invention is not particularly limited as long as it is an oligonucleotide usually used in this field, and a commercially available oligonucleotide or an oligonucleotide appropriately synthesized by a method known per se may be used.
• the oligonucleotide also includes a phosphorothioate-type oligonucleotide. Further, the oligonucleotide in which the 5'end of the "phosphate ester or thiophosphate ester" obtained by the synthetic method of the present invention is deprotected may be used as the oligonucleotide.
• the oligonucleotide in the step (1) in the synthetic method of the present invention is one in which about 1 to 50 nucleosides are bound via a phosphodiester bond or a phosphorothioate bond at the 3'and 5'positions, and the oligonucleotide is at the 3'end. It is preferable that the hydroxy group is protected by a protecting group.
• Examples of the nucleoside include the same nucleosides as those in the step (1) in the synthetic method of the present invention described above, except that the 3'-OH group is not protected, and the preferred nucleosides are also the same.
• the plurality of nucleosides in the oligonucleotide may be the same or different.
• Examples of the protecting group at the 3'end of the oligonucleotide include the same protecting group as the 3'-OH group of the nucleoside in the step (1) in the synthetic method of the present invention described above, and the same is also preferable. ..
• the nucleoside and oligonucleotide in the step (1) in the synthesis method of the present invention may be bound on a carrier for solid-phase synthesis.
• the carrier for solid-phase synthesis is not particularly limited as long as it is usually used in this field, and specifically, for example, porous glass, porous synthetic polymer (for example, polystyrene, polyacrylamide, etc.), silica particles, etc.
• the surface of the above is coated with the resin.
• the amount of phosphoramidite used in step (1) in the synthetic method of the present invention is usually 1 to 5 equivalents with respect to 1 mol of nucleoside or oligonucleotide.
• the content of 5-benzylthio-1H-tetrazole is usually 1 mol with respect to 1 mol of phosphoramidite. It is an amount that is 1 to 20 equivalents.
• the step (1) in the synthesis method of the present invention is not particularly limited as long as the reaction conditions (temperature, pressure, atmosphere, etc.) allow the formation reaction of the phosphite ester to proceed smoothly, and is usually 10 to 40 ° C. It may be carried out in a pressure and inert gas atmosphere.
• the reaction time of step (1) in the synthesis method of the present invention cannot be unequivocally stated because it can change depending on the reaction conditions, but it is usually 1 minute to 3 hours.
• step (2) in the synthesis method of the present invention the phosphite ester, which is the product of step (1), is oxidized with an oxidizing agent to form a phosphoric acid ester, or sulfided with a sulfide agent to produce a thiophosphate ester. This is the process of generating.
• the oxidizing agent is not particularly limited as long as it is usually used in this field, and hydrous iodine is preferable.
• Specific examples include the form of an iodine solution containing water as one of the solvents, and specific examples of the solvent include a mixed solvent of pyridine-tetrahydrofuran-water, a mixed solvent of pyridine-water, and the like. Can be mentioned.
• the concentration of iodine contained in the iodine solution is usually 0.02 to 0.10 mol / L with respect to the total volume of the solution.
• the sulfurizing agent is not particularly limited as long as it is usually used in this field.
• PADS phenylacetyldisulfide
• 3H-1,2-benzodithiol-3-one-1,1 -Dioxide eaucage Reagent
• POS 5-Phenyl-3H-1,2,4-dithiazole-3-one
• DDTT dithiazolin-3-thione
• these may be used in the form of a solution, and specific examples thereof include a POS-containing acetonitrile solution having a concentration of 0.02 to 0.10 mol / L.
• the amount of the oxidizing agent or sulfurizing agent used is usually 1 to 5 equivalents with respect to 1 mol of the phosphite ester.
• the phosphite ester which is the product of the step (1) is specifically.
• a compound represented by the general formula (II-0) can be mentioned.
• Specific examples of the phosphoric acid ester produced in step (2) include compounds represented by the following general formula (II-1), and thiophosphate which is a product of step (2).
• Specific examples of the ester include compounds represented by the following general formula (II-2).
• R 1 represents a group selected from the following functional group group (I-1): (In the functional group group (I-1), n 1 and n 2 each independently represent an integer of 1 to 6.), A 1 represents a nucleoside and A 2 represents a nucleoside or an oligonucleotide. ].
• Examples of the functional group group (I-1) of the general formulas (II-0) to (II-2) include the same as the functional group group (I-1) of the general formula (I), and the preferred ones are also the same. Is.
• nucleosides in A 1 of the general formula (II-0) ⁇ (II -2) the same thing can be mentioned a nucleoside in phosphoramidite in the activation process of the present invention, it is preferable also the same.
• nucleoside in A 2 of the general formulas (II-0) to (II-2) are the same as those in the step (1) in the synthetic method of the present invention described above, and the preferred ones are also the same.
• Examples of the oligonucleotides in A 2 of the general formulas (II-0) to (II-2) are the same as those in the step (1) in the synthetic method of the present invention described above, and the preferred ones are also the same. ..
• the step (2) in the synthesis method of the present invention is not particularly limited as long as the reaction conditions (temperature, pressure, atmosphere, etc.) allow the oxidation reaction or sulfide reaction of the phosphite ester to proceed without delay, and is, for example, 10 to 40. It may be carried out at ° C, normal pressure, and in an inert gas atmosphere.
• the reaction time of step (2) in the synthesis method of the present invention cannot be unequivocally determined because it can change depending on the reaction conditions, but it is usually 1 minute to 3 hours.
• nucleic acid synthesis is usually referred to as (A) nucleoside or oligonucleotide deprotection, (B) coupling reaction, (C) unreacted capping, and (D) coupling product oxidation or sulfide reaction.
• the nucleic acid chain is extended by repeating four steps (reaction cycles).
• the step (1) in the synthesis method of the present invention corresponds to the step (B) in the nucleic acid synthesis
• the step (2) in the synthesis method of the present invention corresponds to the step (D) in the nucleic acid synthesis. Therefore, by repeating the reaction cycle including the synthetic method of the present invention, it is possible to extend the nucleic acid chain and obtain a desired nucleic acid.
• the step (A) in the above nucleic acid synthesis is a step of deprotecting the protecting group at the 5'position of the nucleoside or the oligonucleotide with a deblocking agent.
• the deblocking agent is not particularly limited as long as it is usually used in this field, and a commercially available one or one appropriately synthesized by a method known per se may be used. Specific examples thereof include a dichloroacetic acid-toluene solution and a trichloroacetic acid-dichloromethane solution.
• the step (C) in the nucleic acid synthesis is a step of capping the unreacted product in the step (B) in the nucleic acid synthesis using a capping agent.
• the capping agent is not particularly limited as long as it is usually used in this field, and a commercially available one or one appropriately synthesized by a method known per se may be used. Specifically, for example, a combination of acetic anhydride and N-methylimidazole can be mentioned.
• the steps (A) to (D) in the above-mentioned nucleic acid synthesis are repeated to extend the nucleic acid chain, and then the target nucleic acid is cut out and deprotected from the solid-phase synthesis carrier.
• Ammonia water or a methylamine solution is usually used in this step.
• the ammonia water is not particularly limited as long as it is usually used in this field, and a commercially available one or one appropriately synthesized by a method known per se may be used. Specific examples thereof include an aqueous ammonia solution having a concentration of 25 to 28% by mass, which may be used in the form of a mixed solution of an aqueous solution of methylamine and an aqueous solution of ethanol.
• Example 1 Method for preparing phosphoromidite activator 1 To 29.85 mL of acetonitrile was added 0.15 mL (1.52 mmol) of piperidine to dissolve it, and a 0.5 vol% (volume / volume%) piperidin-added acetonitrile solution was prepared. .. Next, 1.73 g (9.00 mmol) of 5-benzylthio-1H-tetrazole (BTT) (manufactured by Wako Pure Chemical Industries, Ltd.) was added to 30 mL with 0.5 vol% piperidine-added acetonitrile solution, and 0.5 vol% piperidine was added. A 0.30 mol / L BTT phosphoramidite activator 1 containing the above was prepared.
• BTT 5-benzylthio-1H-tetrazole
• Example 2-4 Method for preparing phosphoramidite activator 2-4 Examples except that an acetonitrile solution to which 0.5 vol% of various additives shown in Table 1 was added was used instead of the acetonitrile solution to which 0.5 vol% piperidine was added.
• the phosphoramidite activator 2-4 was prepared in the same manner as in 1.
• Comparative Example 2-12 Method for preparing phosphoramidite activator 102-112 Examples except that an acetonitrile solution to which 0.5 vol% of various additives shown in Table 1 was added was used instead of the acetonitrile solution to which 0.5 vol% piperidine was added.
• the phosphoramidite activator 102-112 was prepared in the same manner as in 1.
• Example 5 Method for preparing phosphoramidite activator 5
• acetonitrile 0.30 mL (3.04 mmol) of piperidine to dissolve it, and a 1.0 vol% piperidine-added acetonitrile solution was prepared.
• 1.73 g (9.00 mmol) of BTT was adjusted to 30 mL with an acetonitrile solution containing 1.0 vol% piperidine to prepare 0.30 mol / L BTT phosphoramidite activator 5 containing 1.0 vol% piperidine.
• Example 6-7 Preparation method of phosphoramidite activator 6-7 Examples except that an acetonitrile solution to which 1.0 vol% of various additives shown in Table 2 was added was used instead of the acetonitrile solution to which 1.0 vol% piperidine was added. A phosphoramidite activator 6-7 was prepared in the same manner as in 5.
• Comparative Example 13-15 Preparation method of phosphoramidite activator 113-115 Examples except that an acetonitrile solution to which 1.0 vol% of various additives shown in Table 2 was added was used instead of the acetonitrile solution to which 1.0 vol% piperidine was added.
• the phosphoramidite activator 113-115 was prepared in the same manner as in 5.
• Example 8-13 Preparation of phosphoramidite activator 8-13 Examples except that the amount of N-methylpiperidine added was 0.5, 0.6, 0.7, 0.8, 0.9, 1.0 vol%, respectively. Phosphoramidite activator 8-13 was prepared in the same manner as in 3.
• Example 14-19 Preparation method of phosphoramidite activator 14-19 Except that the amount of N-methylpyrrolidine added was 0.5, 0.6, 0.7, 0.8, 0.9, 1.0 vol%, respectively. Phosphoramidite activator 14-19 was prepared in the same manner as in Example 4.
• Example 20 Method for preparing phosphoramidite activator 20 BTT 2.02 g (10.5 mmol) was prepared in 30 mL with 0.5 vol% N-methylpiperidine-added acetonitrile solution, and 0.35 containing 0.5 vol% N-methylpiperidine. A mol / L BTT phosphoramidite activator 20 was prepared.
• Example 21-25 Preparation method of phosphoramidite activator 21-25 Example 20 except that the amount of N-methylpiperidine added was 0.6, 0.7, 0.8, 0.9, 1.0 vol%, respectively.
• the phosphoramidite activator 21-25 was prepared in the same manner as above.
• Example 26-31 Method for preparing phosphoramidite activator 26-31 N-methylpyrrolidine was used as an additive instead of N-methylpyrrolidine, and the addition amounts thereof were 0.5, 0.6, 0.7, 0.8, 0.9 and 1.0, respectively.
• the phosphoramidite activator 26-31 was prepared in the same manner as in Example 20 except that it was used so as to be vol%.
• the phosphoramidite activator (0.6 vol% piperidine) that can be stored at a low temperature of 0 to 2 ° C. for 2 weeks , 0.60 vol / L BTT phosphoramidite activator containing 0.6 vol% pyrrolidine, 0.8 vol% N-methylpiperidine, or 0.7 vol% N-methylpyrrolidine) in a liquid phase system Synthesis was performed.
• nucleic acid dimer synthesis was also performed using a 0.30 mol / L BTT phosphoramidite activator containing 0.5 vol% N-methylimidazole and a 0.30 mol / L BTT phosphoramidite activator without additives. , The synthetic results were compared.
• Synthesis Example 1 3', 5'-O-bis (tert-butyldimethylsilyl) thymidin synthesis method Under a nitrogen atmosphere, 25.0 g (103 mmol) of thymidin (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) was added to N, N-. It was dissolved in 75 mL of dimethylformamide (DMF) (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) and 75 mL of tetrahydrofuran (THF) (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) and cooled to 5 ° C.
• DMF dimethylformamide
• THF tetrahydrofuran
• Synthesis Example 2 Synthesis method of 3'-O- (tert- butyldimethylsilyl) thymidine
• the total amount of 3', 5'-O-bis (tert-butyldimethylsilyl) thymidine obtained in Synthesis Example 1 was 194 mL of THF and ions. It was dissolved in 32 mL of exchange water and cooled to 5 ° C. Then, 32 mL of trifluoroacetic acid (TFA) (manufactured by Wako Pure Chemical Industries, Ltd.) was added dropwise to the obtained solution over 20 minutes, and the mixture was stirred at 3 to 5 ° C. for 4 hours.
• THF trifluoroacetic acid
• the pH was adjusted with 450 mL of 1.0 mol / L sodium hydroxide aqueous solution, and the mixture was extracted twice with 300 mL of ethyl acetate.
• the organic layer was washed with 300 mL of saturated brine, sodium sulfate was added, and the mixture was dried. After filtering sodium sulfate, the organic layer was removed under reduced pressure.
• Example 32 Synthesis of nucleic acid dimer using phosphoramidite activator 32 0.6 vol in the same manner as in Example 1 except that the amount of piperidine added was 0.6 vol% instead of 0.5 vol%. A 0.30 mol / L BTT phosphoramidite activator 32 containing% piperidine was prepared.
• Example 32 The reaction scheme of Example 32 is shown below. (In the above reaction scheme, DMTr represents a 4,4'-dimethoxytrityl group and TBS represents a tert-butyldimethylsilyl group.) It was confirmed that the physical characteristic data of the obtained nucleic acid dimer was consistent with the data described in J. Am. Chem. Soc. 2010, 132, 15930.
• Example 33 0.30 mol / containing 0.6 vol% pyrrolidine in the same manner as in Example 32, except that pyrrolidine was used instead of piperidine as a synthetic additive for the nucleic acid dimer using the phosphoramidite activator 33.
• a phosphoramidite activator 33 for L BTT was prepared and used to synthesize a nucleic acid dimer.
• Example 34 Synthesis of Nucleic Acid Dimer Using Phosphoramidite Activator 11 Except that the phosphoramidite activator 11 obtained in Example 11 was used instead of the phosphoramidite activator 32. Nucleic acid dimers were synthesized in the same manner as in Example 32.
• Example 35 Synthesis of Nucleic Acid Dimer Using Phosphoramidite Activator 16 Except that the phosphoramidite activator 16 obtained in Example 16 was used instead of the phosphoramidite activator 32. Nucleic acid dimers were synthesized in the same manner as in Example 32.
• Comparative Example 24 Synthesis of Nucleic Acid Dimer Using Phosphoramidite Activator 101 Except that the phosphoramidite activator 101 obtained in Comparative Example 1 was used instead of the phosphoramidite activator 32. Nucleic acid dimers were synthesized in the same manner as in Example 32.
• Comparative Example 25 Synthesis of Nucleic Acid Dimer Using Phosphoramidite Activator 118 Except that the phosphoramidite activator 118 obtained in Comparative Example 18 was used instead of the phosphoramidite activator 32. Nucleic acid dimers were synthesized in the same manner as in Example 32.
• Table 3 shows the yields calculated from NMR and the isolated yields after purification by silica gel column chromatography in Examples 32-35 and Comparative Examples 24-25.
• the physical characteristic data of the products obtained in Examples 33-35 and Comparative Examples 24-25 were the same as those in Example 32.
• the yield is higher than when the phosphoramidite activator containing no additive is used. It turned out that the reaction proceeded at a rate. Therefore, the phosphoramidite activator of the present invention has a higher storage value than that of the additive (N-methylimidazole) -containing phosphoramidite activator disclosed in Patent Document 1 in liquid phase synthesis. It has been found that it has an excellent effect of having stability and having a high reaction yield equal to or higher than that of a phosphoramidite activator containing no additives.
• the phosphoramidite activator of the present invention in solid phase synthesis, the phosphoramidite activator (0.8 vol% N-methylpiperidine) having good performance in liquid phase synthesis was used.
• DNA oligomer synthesis was carried out in a solid phase system using a 0.30 mol / L BTT phosphoramidite activator containing 0.7 vol% N-methylpyridine.
• DNA oligomer synthesis using a 0.30 mol / L BTT phosphoramidite activator containing 0.5 vol% N-methylimidazole was also performed, and the synthesis results were compared.
• Example 36 Synthesis of DNA oligomer using phosphoramidite activator 11 Universal support (carrier for solid phase synthesis, trade name: Glen Unysupport CPG 1000, manufactured by Glen Research) was applied to the reaction column in an amount equivalent to 1.0 ⁇ mol. Filled with a 0.07M acetonitrile solution of 5'-O- (4,4'-dimethoxytrityl) thymidine-3'-[(2-cyanoethyl) -N, N-diisopropyl] phosphoramidite (manufactured by CARBOSYNTH), and The phosphoramidite activator 11 obtained in Example 11 was prepared, and dT 20mer (5'-TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTT
• Example 37 Synthesis of DNA oligomer using phosphoramidite activator 16 Except that the phosphoramidite activator 16 obtained in Example 16 was used instead of the phosphoramidite activator 11. Similar to Example 36, dT 20mer was synthesized and its purity was measured by HPLC.
• Comparative Example 26 Synthesis of DNA oligomer using phosphoramidite activator 118 Except that the phosphoramidite activator 118 obtained in Comparative Example 18 was used instead of the phosphoramidite activator 11. Similar to Example 36, dT 20mer was synthesized and its purity was measured by HPLC.
• Table 6 shows the purity of dT 20mer measured by HPLC in Examples 36 and 37 and Comparative Example 26.
• RNA oligomer synthesis was carried out in.
• RNA oligomer synthesis using a 0.30 mol / L BTT phosphoramidite activator containing 0.5 vol% N-methylimidazole was also performed, and the synthesis results were compared.
• RNA oligomer using phosphoramidite activator 11 Universal support carrier for solid phase synthesis, trade name: Glen Unysupport CPG 1000, manufactured by Glen Research
• carrier for solid phase synthesis trade name: Glen Unysupport CPG 1000, manufactured by Glen Research
• RNA oligonucleotide was immersed in a mixed solvent of 28% by mass aqueous ammonia-40% by mass methylamine aqueous solution-70% by mass ethanol aqueous solution (1: 1: 1) at 60 ° C. for 3 hours for solid-phase synthesis.
• RU 20mer was excised from the carrier and deprotected.
• the obtained solution is concentrated, 50 ⁇ L of dimethyl sulfoxide and 200 ⁇ L of triethylamine hydrofluorate (TREAT-3HF) are added thereto, and the solution is immersed at 60 ° C. for 3 hours to deprotect the tert-butyldimethylsilyl group. Carried out.
• the purity of the obtained rU 20mer was measured by HPLC under the same measurement conditions as in Example 36.
• Example 39 Synthesis of RNA oligomer using phosphoramidite activator 16 Except that the phosphoramidite activator 16 obtained in Example 16 was used instead of the phosphoramidite activator 11. Similar to Example 38, rU 20mer was synthesized and its purity was measured by HPLC.
• Comparative Example 27 Synthesis of RNA oligomer using phosphoramidite activator 118 Except that the phosphoramidite activator 118 obtained in Comparative Example 18 was used instead of the phosphoramidite activator 11. Similar to Example 38, rU 20mer was synthesized and its purity was measured by HPLC.
• Table 7 shows the purity of rU 20mer measured by HPLC in Examples 38 and 39 and Comparative Example 27.

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